Using proteases to control restriction enzyme activity

ABSTRACT

Proteases are enzymes which hydrolyze protein enzymes, eliminating their activity. The present invention exploits the hydrolyzing activity of proteases including proteinase K, endoproteinase LysC and/or trypsin to control the activity of restriction enzymes and/or eliminate or reduce production of unwanted DNA or RNA fragments (known as star activity).

BACKGROUND

Restriction enzymes, also known as restriction endonucleases, are asubclass of hydrolase enzymes that hydrolyze ester bonds, act as DNA orRNA nucleases and cleave specific DNA or RNA sequences. When performingan enzyme digestion reaction, the activity of all enzymes involvedshould be optimized for precision in obtaining the expected product orresult. Most restriction enzymes catalyze the substrate (DNA or RNA)continuously over time, thus continuously accumulating the desired DNAor RNA fragments and occasionally, unwanted side products.

Though optimization of enzyme reactions with restriction endonucleasesare usually attempted through specific buffer and reaction timeconditions, there are sometimes undesired reactions and relatedproblems, especially when enzyme displays off-target activity inaddition to its main activity. Optimized restriction endonucleaseactivity typically digests a specified amount of substrate to completionwithin a set time. However, some restriction endonucleases generate sidereactions and unwanted DNA or RNA cleavage with accumulated activity,especially with excess enzyme in a reaction, where the reactioncontinues for a long period, under particular buffer conditions, orwhere there is high glycerol. Such cleavage at sites other than thecognate restriction sites is known as star activity.

Controlling the concentration of enzyme or the overall reaction time,such as terminating the reaction via heat inactivation or otherpurification method when the reaction is completed, is a common methodto limit total enzyme activity and thus reduce star activity. Othermethods of controlling the reaction may be more desirable, particularlywhere fine control to achieve a higher reliability of the assay isdesired.

SUMMARY

Proteases are enzymes which hydrolyze protein enzymes, eliminating theiractivity. The present invention exploits the hydrolyzing activity ofproteases to control the activity of restriction enzymes and eliminateor reduce production of unwanted DNA or RNA fragments.

In this invention, proteases are initially included with or added atsome point to the restriction endonuclease reaction mix, such that therestriction enzymes are inactivated by the protease during the course oftheir DNA/RNA digestion reaction. In this manner, total enzyme activityis controlled not only by buffers and reaction times, but also by therelative quantities of substrate, restriction endonucleases andproteases (and sometimes, including the protease addition time) in orderto further limit over-digestion and star activity. Protease inhibitorscan optionally be used to stop or inhibit protease activity at a desiredpoint in the reaction.

The invention is further described in the figures and description whichfollow, including in the examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Theoretical complete pattern of BamHI, EcoRI and NcoI on lambdaDNA according to the DNA sequence and enzyme recognition and cuttingspecificities. The pattern can be aligned with any of the bands shown inFIGS. 3-8 to indicate completion of cleavage and star activity.

FIG. 2: Panel A: Predicted intrinsic restriction endonuclease activity.Solid line: restriction endonuclease activity without proteases; dashedline, restriction endonucleases with proteases. Panel B: Totalrestriction endonucleases activity. Solid line: total restriction enzymeactivity without proteases; dashed line, total restriction enzymeactivity with proteases.

FIG. 3: The enzyme activity of EcoRI on lambda DNA in buffers A1 andA1S2. Panel A: 1 hour in buffer A1, Panel B: 1 hour in buffer A1S2,Panel C: 20 hours in buffer A1, Panel D: 20 hours in buffer A1S2. Thedescending triangle (top) represents the 2-fold serial dilution of therestriction endonuclease.

FIG. 4: The enzyme activity of EcoRI on lambda DNA in buffers A2 andA2S2. Panel A: 1 hour in buffer A2, Panel B: 1 hour in buffer A2S2,Panel C: 20 hours in buffer A2, Panel D: 20 hours in buffer A2S2. Thedescending triangle (top) represents the 2-fold serial dilution of therestriction endonuclease.

FIG. 5: The enzyme activity of EcoRI on lambda DNA in buffers A3 andA3S2. Panel A: 1 hour in buffer A3, Panel B: 1 hour in buffer A3S2,Panel C: 20 hours in buffer A3, Panel D: 20 hours in buffer A3S2. Thedescending triangle (top) represents the 2-fold serial dilution of therestriction endonuclease.

FIG. 6: The enzyme activity of BamHI on lambda DNA in buffers A3 andA3S2. Panel A: 1 hour in buffer A3, Panel B: 1 hour in buffer A3S2,Panel C: 20 hours in buffer A3, Panel D: 20 hours in buffer A3S2. Thedescending triangle (top) represents the 2-fold serial dilution of therestriction endonuclease.

FIG. 7: The enzyme activity of NcoI on lambda DNA in buffers A3 andA3S2. Panel A: 1 hour in buffer A3, Panel B: 1 hour in buffer A3S2,Panel C: 20 hours in buffer A3, Panel D: 20 hours in buffer A3S2. Thedescending triangle (top) represents the 2-fold serial dilution of therestriction endonuclease.

FIG. 8: The results of enzyme activity of EcoRI on lambda DNA. Panel A:1 hour in buffer A2LysC, Panel B: 1 hour in buffer A2Trypsin, Panel C:22 hours in buffer A2LysC, Panel D: 22 hours in buffer A3Trpsin. Thedescending triangle (top) represents the 2-fold serial dilution of therestriction endonuclease.

FIG. 9: Flow chart showing automated genotyping/sequencing/alleletyping/gene expression analysis using the reagents and techniques of theinvention.

FIG. 10: Flow chart showing optimization of a restriction enzymereaction where a protease is used to control it.

DETAILED DESCRIPTION

Restriction enzymes which can be used in the invention include bothendonucleases and catalytic RNAs (ribozyes), DNases and RNases. DNAasesinclude a large number of enzymes which, based on their targets forcleavage, are further classified into Type I, Type II (where BamHI,EcoRI and NcoI in the examples below are Type IIP; palindromic), TypeIII, Type IV and Type V (e.g., the cas9-gRNA complex from CRISPR) aswell as artificial restriction enzymes, which can target large DNA sites(up to 36 bp) and can be engineered to bind to desired DNA sequences.Zinc finger nucleases are the most commonly used artificial restrictionenzymes.

Proteases can be classified into endopeptidases, which cleave the targetprotein internally, and exopeptidases—where carboxypeptidases digestamino acids from the carboxy-terminal end of a protein, andaminopeptidases digest proteins from the amino-terminus.

Proteases can further be classified based into seven broad groups, basedon the catalytic residue used to facilitate proteolysis: serineproteases (using a serine alcohol as the catalytic residue); cysteineproteases (using a cysteine thiol as the catalytic residue); threonineproteases (using a threonine secondary alcohol as the catalyticresidue); aspartic proteases (using an aspartate carboxylic acid as thecatalytic residue); glutamic proteases (using a glutamate carboxylicacid as the catalytic residue); metalloproteases (using a metal, usuallyzinc, for catalysis); and, asparagine peptide lyases—using an asparagineto perform an elimination reaction (not requiring water).

Any of the foregoing proteases which prove suitable for controllingrestriction enzyme reactions, including in order to reduce star activity(in addition to those in the examples below) can be used in theinvention. Specific examples of proteases include: TEV protease, whichis specific for the sequence ENLYFQ \S; trypsinogen; carboxypeptidase B;enterokinase; Lys-C and Thrombin.

Instead of including protease in the reaction mixture with therestriction endonuclease and the substrate, one can add the protease ata later time in the reaction process to inhibit or arrest nucleaseactivity. The timing of adding the components can also be optimized withroutine experimentation.

Protease inhibitors include alpha 1-antitrypsin, alpha1-antichymotrypsin, C1-inhibitor, antithrombin, plasminogen activatorinhibitor-1, and neuroserpin. To control the protease portion of thereaction with an inhibitor, the inhibitor would need to selected so asto be one which inhibited the protease being used in the reaction todigest the restriction enzyme(s) used in the reaction. The proteaseinhibitor could be included with the reaction mixture or added at alater time, and it's addition would need to be optimized as part ofoptimization of the reaction process.

Specific examples of DNA-cleaving restriction endonucleases which can beused with the process of the invention (other than those in the examplesbelow) include: HindIII, HincII, MutH, ExoRII, EcoR124I, EcoRV, EcoP151,Dnase1, Nuclease S1, Bal 31, DNAQ, Taq1, Not1, TREX1, TREX2 and WRNexonuclease. Specific examples of RNA-cleaving restriction nucleaseswhich can be used in cleaving RNA with the process of the inventioninclude endoribonucleases such as RNase III, RNase A, RNase H, RNase L,and RNase P; and exoribonucleases such as PNPase, RNase D, RNase R, andRNase T.

Among DNA restriction endonucleases which cleave at a unique recognitionsequence: BamHI recognizes and cuts at G/ GATCC; EcoRI recognizes andcuts at G/ AATTC; and NcoI recognizes and cuts at C/ CATGG. FIG. 1 showsthe theoretical complete digestion pattern of BamHI, EcoRI and NcoI onlambda DNA, followed by gel electrophoresis. These three enzymes areused in the examples below, and the bands they generate in the examplescan be compared to FIG. 1 for reference. Star activity is shown in FIGS.3 to 8 (if present) as extra bands on the DNA gel in the left lanes(which represent high enzyme concentration).

EXAMPLE 1

Schematic depiction of the enzymatic effect on substrate is shown inFIG. 2. In this example, the restriction endonuclease has very stableactivity, maintaining 100% activity over time. Conversely, addition ofprotease, in this example, reduces restriction endonuclease activity by10% per unit of time (i.e. per minute). After 10 time-units, there is noactive restriction endonuclease remaining, as shown in Panel A. Theaccumulated restriction endonuclease activity is shown in panel B. Whilethe total product of enzyme activity keeps increasing for therestriction endonuclease without protease, the reaction containingprotease accumulates a limited amount of total activity, and thenceases.

In a formula to represent the activity of a stable restrictionendonuclease, without protease, restriction endonuclease activity(RE_(A)) is consistently at 1.RE_(A)=1

Total enzyme activity (RE_(T)) is determined over a time, designatedwith units T. With protease, restriction endonuclease activity (RE_(P))is 1−0.1 T when T is no more than 10, and zero when T is larger than 10.RE _(P)=1−0.1T (when T is no more than 10)

The total activity (RE_(T)) is 0.5 T−0.05T² when T is no more than 10,and a constant 5 after T is larger than 10.RE _(T)=0.5T−0.05T ² (when T is no more than 10)

Since the simultaneous usage of protease controls the total output ofthe restriction endonucleases, it is referred to hereinafter as theinternal protease control.

Example 2: The Internal Protease Control is Independent of RestrictionEnzyme Reaction Buffers

In this example, restriction endonuclease EcoRI, Diluent C, proteinaseK, 6× STOP solution, and lambda DNA (substrate) were purchased from NewEngland Biolabs. EcoRI is a highly active restriction endonuclease,which under certain circumstances (i.e. specific buffers) can havecleave at sites other than its cognate restriction sites, which is knownas star activity. Proteinase K has broad specificity, predominantlycutting the peptide bonds adjacent to hydrophobic amino acids, and isactive in wide range of buffers, so it can retain activity in a varietyof reaction conditions including all restriction enzyme buffers, as longas there is no protease inhibitors

Three different reaction buffers were tested in this example, with andwithout the addition of protease:

Buffer A1: 20 mM Tris-HCl, pH 7.4, 40 mM KoAc, 10 mM MgCl₂, 0.1 mM CaCl₂

Buffer A2: 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mM MgCl₂, 0.1 mM CaCl₂

Buffer A3: 20 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 0.1 mM CaCl₂

Buffer A1S2: Buffer 1 plus 0.05 units/ml proteinase K

Buffer A2S2: Buffer 2 plus 0.05 units/ml proteinase K

Buffer A3S2: Buffer 3 plus 0.05 units/ml proteinase K.

EcoRI was serially diluted using Diluent C in 2-fold dilutions (whereeach successive lane in the figures represents a successive 2-folddilution relative to the preceding lane). Digests of lambda DNA wereperformed in Buffer A1, Buffer A2, Buffer A3, and Buffer A1S2, BufferA2S2, and Buffer A3S2. Reactions were stopped by adding 6× STOP solutionat 1 hour or 20 hours.

In FIG. 3 using buffer A1: EcoRI can digest lambda DNA to completion atthe 8^(th) lane but shows star activity between the 1^(st) to 3^(rd)lane at 1 hour. EcoRI can digest lambda DNA to completion through the11^(th) lane but has star activity between the 1^(st) to 6^(th) lanes at20 hours.

In FIG. 3 using Buffer A1S2: EcoRI can digest lambda DNA to completionthrough the 3^(rd) lane and has no star activity in any lanes at 1 hour.EcoRI can digest lambda DNA to completion through the 4^(th) lane andhas no star activity in any lanes at 20 hours.

In FIG. 4 using Buffer A2: EcoRI can digest lambda DNA to completionthrough the 9^(th) lane but has star activity in the 1^(st) lane at 1hour. EcoRI can digest lambda DNA to completion through the 12^(th) lanebut shows star activity between the 1^(st) to 5^(th) lanes at 20 hours.

In FIG. 4 using Buffer A2S2, EcoRI can digest lambda DNA to completionthrough the 5^(th) lane but has no star activity in any lanes at 1 hour.EcoRI can digest lambda DNA to completion through the 6^(th) lane butshows no star activity in any lanes at 20 hours.

In FIG. 5 using Buffer A3, EcoRI can digest lambda DNA to completionthrough the 7^(th) lane but shows no star activity at 1 hour. EcoRI candigest lambda DNA to completion through the 9^(th) lane, but has staractivity between the 1^(st) to 4^(th) lanes, at 20 hours.

In FIG. 5 using Buffer A3S2, EcoRI can digest lambda DNA to completionthrough the 6^(th) lane but has no star activity in any lanes at 1 hour.EcoRI can digest lambda DNA to completion through the 6^(th) lane buthas no star activity in any lanes at 20 hours.

The results shown in FIGS. 3-5 show that addition of proteinase Kchanged the performance of EcoRI in all buffers A1, A2 and A3, whichwere re-named as buffer A1S2, A2S2, A3S2 respectively, when proteinase Kwas included.

Example 3: The Internal Protease Control Works with any RestrictionEndonuclease

All restriction endonucleases are proteins, and all proteins can bedigested by proteinase K, thus the process is expected to be aseffective on other restriction endonucleases as with EcoRI.

In addition to the EcoRI in Buffer A3 and A3S2 (see Example 2) two otherrestriction endonucleases were also tested: BamHI and NcoI (from NewEngland Biolabs, Inc.)

BamHI and NcoI were each diluted with Diluent A (New England Biolabs,Inc.) in a 2-fold serial dilution, where the substrate was lambda DNA,and the buffers were Buffer A3 or A3S2. The reactions were stopped byadding 6× STOP solution at 1 hour and 20 hours respectively.

In FIG. 6 using Buffer A3: BamHI can digest lambda DNA to completionthrough the 7^(th) lane and has no star activity at 1 hour. BamHI candigest lambda DNA to completion through the 9^(th) lane but displaysstar activity between the 1^(st) to 4^(th) lanes at 20 hours.

In FIG. 6 using Buffer A3S2: BamHI can digest lambda DNA to completionthrough the 6^(th) lane and has no star activity at 1 hour. BamHI candigest lambda DNA to completion through the 6^(th) lane and has no staractivity in any lanes at 20 hours.

In FIG. 7 using Buffer A3: NcoI can digest lambda DNA to completionthrough the 8^(th) lane and has no star activity at 1 hour. NcoI digestslambda DNA to completion at 14^(th) lane and has star activity betweenthe 1^(st) to 5^(th) lanes at 20 hours.

In FIG. 7 using Buffer A3S2, NcoI can digest lambda DNA to completionthrough the 8^(th) lane and has no star activity in any lanes at 1 hour.NcoI can digest lambda DNA to completion through the 13^(th) lane buthas no star activity in any lanes at 20 hours.

This example shows that in addition to EcoRI in Example 2, desirableenzyme activity of BamHI and NcoI can be changed by the addition ofproteinase K.

Example 4: Internal Protease Control using a Protease other thanProteinase K

Proteinase K has a broad specificity for protein digestion and is activein most buffers. Other proteases, if active in the specific reactionbuffer of the enzyme for the primary reaction, can substitute forproteinase K as the internal protease control.

Endoproteinase LysC is a serine endoproteinase that cleaves peptidebonds at the carboxyl side of lysine, and Trypsin is serineendoproteinase that cleaves peptide bonds at the carboxyl side of lysineor arginine. Both enzymes were purchased from New England Biolabs.

Buffer A2LysC: Buffer A2 plus 400 ng/ml endoproteinase LysC.

Buffer A2Trypsin: Buffer A2 plus 25 ng/ml trypsin.

EcoRI was diluted with Diluent C in a serial 2-fold dilution, thesubstrate was lambda DNA, and digestion was performed in Buffer A2LysCor Buffer A2Trpsin. The reaction was stopped by adding 6× STOP solutionat 1 hour and 22 hours respectively.

In FIG. 8 using Buffer A2LysC: EcoRI can complete the digestion oflambda DNA through the 5^(th) lane at 1 hour with no star activity andcan complete the digestion of lambda DNA through the 4^(th) lane at 22hours with no star activity. Using buffer A2Trpsin: EcoRI can completethe digestion of lambda DNA at 6^(th) lane at 1 hour with no staractivity and can complete the digestion of lambda DNA at 6^(th) lane at22 hours with no star activity.

This example shows that other proteinases capable of digestingrestriction endonucleases can be used as the internal protease controlfor restriction endonucleases activity.

Applications for the Process of the Invention

The uses for the process of the invention include any application wherenucleic acids are identified through their fragmentation patterns ascaptured using gel electrophoresis, and including DNA or RNA sequencingor genotyping, or allele typing, employing such identification means.Specific applications include biometric identification of tissue, bloodor body fluid (living or not), or other sample donor identification ine.g., forensics or therapy; determination of ancestral information, andother research applications where a high degree of reliability is neededin identifying a nucleic acid source or its sequence. The process of theinvention could also be used in gene expression analysis, whereincreased gene expression would present in the gel results as certainthicker and/or darker bands.

One method of automated sequencing, genotyping, allele typing or geneexpression analysis of a sample using the reagents and processes of theinvention is set forth in flow chart form in FIG. 9. The steps in FIG. 9could be automatically performed (using e.g., a robot, which addsreagents at timed intervals from particular containers). In the laststep, the gel results are automatically read and processed by a centralprocessor capable of correlating them with known results, in order toidentify the source of the sample or indicate the likely ancestry of thesample donor (based on presence of certain fragments followingcleavage).

Optimization of Reaction

A general method for determining the proteases to use with particularrestriction endonucleases, other than those exemplified below, is toperform the experiments set forth below in the examples with othercombinations of proteases and restriction endonucleases, and withappropriate buffers, and perform the activity assay described andexemplified herein, with an appropriate level of serial dilution ofrestriction endonucleases as herein, to find the combinations ofcomponents which are best suited for particular functions, includingfinding those with attributes like most active per unit of restrictionendonucleases and protease, and which can most effectively minimize staractivity.

Certain restriction endonucleases or certain types thereof, are neededor preferred for certain functions, and those can be combined withcompatible proteases, in compatible proportions and in appropriatebuffers, all of which can be determined by routine experimentationfollowing the same methods as used to find the combinations set forth inthe examples below.

Other types of restriction enzymes which can be used in the inventioninclude both protein endonucleases and catalytic RNAs (ribozyes), andboth DNases and RNases. Similar experiments to those described hereincan be performed to find suitable RNase/protease combinations forcontrolling RNase reactions and/or reducing star activity in suchRNA-digesting reactions. When using an unknown restriction enzyme, RE,and one wishes to use protease to control the reaction or inhibit oreliminate star activity, the steps in the flow chart of FIG. 10 can befollowed to optimize the reaction conditions. The steps in FIG. 10 trackthose set forth in FIGS. 3 to 8, which were used to determine theoptimal reaction conditions for the restriction enzymes set forth there.One significant step is to perform a serial dilution to determine theoptimal concentrations in the reaction mixture. The steps in FIG. 10 canbe performed with each restriction enzyme, RE, one wishes to determineoptimal conditions for use.

The specific processes, methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “including”, containing”, etc. are to beread expansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims.

It is also noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference, and theplural include singular forms, unless the context clearly dictatesotherwise. Under no circumstances may the patent be interpreted to belimited to the specific examples or embodiments or methods specificallydisclosed herein. Under no circumstances may the patent be interpretedto be limited by any statement made by any Examiner or any otherofficial or employee of the Patent and Trademark Office unless suchstatement is specifically and without qualification or reservationexpressly adopted in a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intent in the use of such terms andexpressions to exclude any equivalent of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention as claimed.Thus, it will be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

What is claimed is:
 1. A process of inhibiting or preventing staractivity by NcoI or BamHI comprising: digesting an oligomer using areaction mixture including NcoI or BamHI, buffer and oligomer; includingin the reaction mixture or adding proteinase K, endoproteinase LysCand/or trypsin in order to inhibit or prevent star activity by NcoI orBamHI; and terminating the digestion by adding a stopping solutionincluding a loading dye.
 2. The process of claim 1 wherein the oligomeris lambda DNA.
 3. The process of claim 1 wherein the buffer is 20 mMTris-HCl, pH 7.4, 40 mM KoAc, 10 mM MgCl₂, 0.1 mM CaCl₂; or 20 mMTris-HCl, pH 7.5, 40 mM NaCl, 10 mM MgCl₂, 0.1 mM CaCl₂.
 4. The processof claim 1 wherein the stopping solution includes 2.5% Ficoll®-400, 11mM EDTA (pH 8.0), 3.3 mM Tris-HCL, 0.017% SDS, 0.015% bromophenol blue.5. The process of claim 4 wherein the stopping solution is added.
 6. Aprocess of determining the optimal relative concentrations to inhibit orprevent star activity by Ncol or BamHI, or Ncol or BamHI and aproteinase comprising: (i) combining Ncol or BamHI, the proteinase, abuffer and an oligomer in order to achieve a starting concentration ofeach; (ii) determining the star activity of Ncol or BamHI after areaction time, and if there is any, diluting the reaction mixture andrepeating the reaction for said reaction time; (iii) repeating step (ii)until there is no star activity; and (iv) determining the concentrationsof Ncol or BamHI and proteinase at which star activity ceases.
 7. Theprocess of claim 6 wherein the proteinase are combined simultaneouslywith the restriction endonuclease.
 8. The process of claim 6 wherein theproteinase is proteinase K, endoproteinase LysC or trypsin.
 9. Theprocess of claim 6 wherein the oligomer is lambda DNA.
 10. The processof claim 6 wherein the buffer is: 20 mM Tris-HCl, pH 7.4, 40 mM KoAc, 10mM MgCl2, 0.1 mM CaCl2; or 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mMMgCl2, 0.1 mM CaCl2; and, 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mMCaCl2.
 11. The process of claim 6 wherein Ncol or BamHI activity isterminated by addition of STOP solution.
 12. The process of claim 1wherein the buffer is 20 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 0.1 mM CaCl₂.